4‑Phenylaminomethyl-Benzeneboric Acid Modified Tip Extraction for Determination of Brassinosteroids in Plant Tissues by Stable Isotope Labeling–Liquid Chromatography–Mass Spectrometry

Abstract

Monitoring brassinosteroids (BRs) has been of major interest of researchers as these substances play a crucial role in a variety of phytological processes in plants. However, the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues. In this study, a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis. In the proposed strategy, a mixed mode sorbent (C8-SO₃H) in tip was modified with 4-phenylaminomethyl-benzeneboric acid (4-PAMBA) through cation exchange and hydrophobic interactions, and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boric acid groups of 4-PAMBA and cis-diol on BRs. The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO₃H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile, followed by LC-MS/MS analysis. BR standards, isotopically labeled with <i>d</i>₅-4-phenylaminomethyl-benzeneboric acid (4-PAMBA-<i>d</i>₅) were introduced to improve the assay precision of LC-MS/MS. Under the optimized conditions, the overall process could be completed within 1 h, which is greatly improved in speed compared with previously reported protocols. In addition, the detection sensitivities of labeled BRs were improved by over 2000-fold compared with unlabeled BRs, thus the consumption of plant materials was reduced to 50 mg. Finally, the proposed method was applied for the investigation of BRs response in rice toward Cd stress